Effective depletion of alloreactive lymphocytes from peripheral blood mononuclear cell preparations

Background. T cells present in an allogeneic bone marrow transplant may produce graft-versus-host disease but also contribute to immune reconstitution and enhance engraftment. Our aim was to separate alloreactive from nonalloreactive T lymphocytes, by performing a mixed lymphocyte culture (MLC) stimulation of donor cells, followed by selective depletion of activated cells expressing the high-affinity interleukin 2 receptor. We then characterized the resulting depleted cell fraction. Methods. Donor peripheral blood mononuclear cells were cocultured with irradiated peripheral blood mononuclear cells from HLA-nonidentical recipient stimulators in an MLC. After 3 days, CD25⁺ lymphocytes (alloreactive cells expressing the alpha chain of the interleukin 2 receptor) were removed by immunomagnetic separation. The depleted donor fraction and untreated cells were then rechallenged in a secondary MLC with the original irradiated stimulator cells or a third party to assess relative alloreactivity. Results. Inhibition of the secondary MLC and of host-specific cytotoxic activities was observed as well as a disappearance of interleukin 2 receptor-positive cells. Alloreactivity against unrelated third-party cells was preserved. Limiting dilution analysis of residual alloantigen-reactive T lymphocytes demonstrated a 1.3 log reduction of antihost reactivity. The depletion largely removed host-specific alloreactive CD4+ cells. Conclusions. This method reduces alloreactivity while retaining reactivity against third-party targets. This approach may allow therapeutic infusion of T cells after HLA-nonidentical allografts with a reduced capacity to produce graft-versus-host disease.